This invention relates to methods for propagating fastidious human viruses in the laboratory, and producing purified viral suspensions of such viruses. It also relates to methods of determining the infectivity of samples containing these viruses and assays for determining the antiviral activity of substances.
The scientific study of many disease-causing human viruses is severely hampered by the inability to grow or propagate them in vitro or in laboratory animals at all or in sufficient numbers to subject them to analytical techniques. Thus, little is known about their characteristics and the means by which they cause disease. In addition, the inability to obtain a sufficient amount of infectious virus particles at a sufficient degree of purity prevents the development of assays for the detection of the presence of the virus in samples from patients. This inability also prevents the development of vaccines or drugs to prevent or treat the diseases caused by these viruses. Thus, in many respects, the ability to make significant medical progress against a number of virus-caused illnesses in humans will depend upon the development of techniques for propagating these viruses in the laboratory.
Because of the inability or difficulty in propagating these viruses in the laboratory, they are known as fastidious viruses. Examples of such fastidious viruses include varicella-zoster virus (VZV), cytomegalovirus, various gastroenteritis viruses, and the human papillomaviruses.
VZV produces varicella (or chicken pox) and zoster. Chicken pox is a common and generally mild childhood disease. However, in adults and in immunosuppressed individuals, it can cause serious and even fatal illness. Zoster is a disease often found in adults and involves lesions similar to chicken pox. It is the manifestation of the reactivation of an earlier varicella infection. It can lead to a number of complications and can be particularly serious for immunosuppressed patients. See Joklik et al., Zinsser Microbiology, 17th Ed. (Appleton-Century Crofts, New York, 1980), pp. 1002-1005.
Humans are apparently the only natural host for VZV. Although VZV has been grown in tissue culture, it remains cell associated and is rapidly inactivated when the host cell is disrupted.
Cytomegalovirus, which is related to VZV in that both are members of the herpesvirus family, is responsible for a broad spectrum of disease affecting humans in many different clinical settings. It is the most common cause of congenital viral infection, it is an etiologic agent of mononucleosis, and is a life-threatening pathogen in immuno-compromised patients. See Joklik et al., Zinsser Microbiology, 17th Ed. (Appleton-Century Crofts, New York, 1980), pp. 1005-1008.
A number of viruses are implicated in acute gastroenteritis, a problem encountered daily by physicians. In developed countries, acute gastroenteritis is second only to the common cold in frequency of occurrence. The illness can be serious and even life-threatening in the elderly, the young, and the debilitated patient. The two most frequently implicated viruses are rotaviruses and the Norwalk virus (and Norwalk-like viruses).
These and other suspected agents of human viral gastroenteritis have been very fastidious with respect to cultivation by in vitro techniques or in laboratory animal hosts. Thus, the studies of these agents have been hampered, and current methods of detecting their presence leave much to be desired. Similarly, vaccine development and methods of prevention and treatment have been extremely hampered. The cultivation of these viral agents will facilitate the development of diagnostic reagents and the development and evaluation of vaccines. See Cukor and Blacklow, "Human Viral Gastroenteritis," Microbiological Reviews, 48:157-179 (1984); Barnett, "Viral Gastroenteritis," Medical Clinics of North America. 67:1031-1058 (1983); and Murphy, "Aetiology of Viral Gastroenteritis: A Review," Med. J. Aust, 2:177-182 (1981).
The human papillomaviruses are a heterogeneous group of viruses that induce epithelial or fibroepithelial proliferations of skin or mucosa. Over 40 types of human papillomavirus (HPV) are recognized, many of which are associated with distinctive lesions. Specific diseases associated with HPV infection, such as common warts, epidermodysplasia verruciformis, and genital warts (condylomas), correlate with specific HPV types. For example, HPV-1 has been shown to produce skin warts, and HPV-11 has been shown to produce genital warts. The latter type can also produce warts on the vocal cords of newborns who have been infected by their mothers. Such warts are a serious problem to the newborns because they threaten breathing and must be surgically removed.
Recent studies have implicated HPVs in the development of premalignant and malignant lesions of the skin (Ikenberg et al., Int. J. Cancer, 32:563-565, 1983; Orth et al., Cancer Res., 39:1074-1082, 1979), uterine cervix (Durst et al., Proc. Natl. Acad. Sci., 80:3812-3815, 1983), and larynx (Galloway et al., Arch. Otol., 72:289-294, 1960). For example, HPV-16 and HPV-18 has been isolated and molecularly cloned from cervical carcinoma cells and are strongly associated with cervical carcinomas. However, the biological significance of the association of HPV-16 and HPV-18 DNA with cervical cancer has not yet been determined.
Studies on the contribution of HPVs to the etiology of human tumors have been severely restricted by the unavailability of laboratory animal hosts or culture systems that would allow neoplastic transformation and/or viral replication. Many unsuccessful attempts have been made to develop tissue culture systems or find laboratory animal hosts for HPV. However, these viruses are fastidious in their growth requirements, being host and cell specific and requiring an epithelial cell in an advanced state of differentiation for replication. HPV infection of human subjects is, of course, ethically unacceptable.
With respect to attempts to grow HPV in tissue culture, recent studies report only that HPV DNA can persist in and transform cultured cells. See La Porta et al., Proc. Natl. Acad. Sci. U.S.A., 79: 3393-3397, 1982, Watts et al., Science, 225:634-636, 1984, and Yasumoto et al., J. Virology, 57:572-577, 1986. The replication of the viruses themselves has not been reported.
Several unsuccessful attempts have been made to develop systems in which HPV-infected human tissues were grafted to immunologically privileged sites on laboratory animals or to immunologically deficient animals. Kreider et al., J. Invest. Dermatology, 57:66-71, 1971 reported that human skin infected with HPV and transplanted to the cheekpouch of hamsters, which is an immunologically privileged site that accepts grafts of foreign tissues, did not produce papillomas after 14 weeks. Pass et al., J. Invest. Dermatology, 61:371-374, 1973 reported that human skin grafts that had been inoculated with an extract of verrucae vulgaris and transplanted to immunosuppressed mice produced no papillomas after 14 weeks. Similarly, Cubie, British J. Dermatology, 94: 659-665, 1976 reported that human skin infected by HPV and grafted onto nude mice showed no papillomas after 9 weeks.
The inventors undertook to develop a method of propagating fastidious human viruses in a laboratory animal. Such a method would allow the production of a large, constant supply of these viruses for further study and testing of their association with human diseases. It would also allow the purification of the virus so that it might be used, for example, in diagnostic agents for the detection of the virus and in vaccines for the prevention of diseases caused by fastidious human viruses.